High serum C-X-C motif chemokine ligand 10 (CXCL10) levels may be associated with new onset interstitial lung disease in patients with systemic sclerosis: evidence from observational, clinical, transcriptomic and in vitro studies

Summary Background Systemic sclerosis-interstitial lung disease (SSc-ILD) is the leading cause of death in patients with SSc. There is an unmet need for predictive biomarkers to identify patients with SSc at risk of ILD. Previous studies have shown that interferon (IFN) pathways may play a role in SSc. We assessed the use of C-X-C motif chemokine ligand 10 (CXCL10) as a predictive biomarker for new onset of ILD in patients with SSc. Methods One-hundred-sixty-five (Female, N = 130) patients with SSc (SSc-ILD, N = 41) and 13 (Female, N = 8) healthy controls were investigated retrospectively. CXCL10 protein levels were measured by ELISA. We performed log rank analysis with baseline CXCL10 serum levels. CXCL10 nanoString data from lung tissues obtained from transplanted patients with SSc-ILD were extracted. Fifteen (Female, N = 10) patients with SSc (SSc-ILD, N = 7) were recruited for bronchoalveolar lavage (BAL) procedure. Lung fibroblasts were treated with BAL-fluid or serum from patients with SSc with or without ILD. Inflammatory/fibrotic genes were assessed. Findings Serum CXCL10 levels were higher in patients with SSc-ILD compared to SSc patients without ILD [Median (IQR):126 pg/ml (66–282.5) vs. 78.5 pg/ml (50–122), P = 0.029, 95% CI: 1.5 × 10−6 to 0.4284]. Survival analysis showed that baseline CXCL10 levels >78.5 pg/ml have a 2.74-fold increased risk of developing new onset of ILD (Log-rank: P = 0.119) on follow-up. CXCL10 levels in BAL supernatant were not different in patients with SSc-ILD compared to SSc without ILD [76.1 pg/ml (7.2–120.8) vs. 22.3 pg/ml (12.1–43.7), P = 0.24, 95% CI: −19.5 to 100]. NanoString showed that CXCL10 mRNA expression was higher in inflammatory compared to fibrotic lung tissues [4.7 (4.2–5.6) vs. 4.3 (3.6–4.7), P = 0.029]. Fibroblasts treated with SSc-ILD serum or BAL fluids overexpressed CXCL10. Interpretations Clinical, transcriptomic, and in vitro data showed that CXCL10 is potentially involved in early SSc-ILD. More research is needed to confirm whether CXCL10 can be classified as a prospective biomarker to detect patients with SSc at higher risk of developing new onset ILD. Funding This collaborative project is co-financed by the 10.13039/501100016238Ministry of Economic Affairs and Climate Policy of the Netherlands utilizing the PPP-allowance made available by the 10.13039/100016036Top Sector Life Sciences & Health to stimulate public-private partnerships (PPP-2019_007). Part of this study is financially supported by 10.13039/100013995Sanofi Genzyme (NL8921).


PROTOCOL SIGNATURE SHEET
The undersigned (Principal) investigator and head of department UMCG confirm that the study and its procedures will comply with the present study protocol and the nWMO Kaderreglement UMCG.Without ethical approval the data/biomaterials will not be used for other (research) purposes (e.g.'FAIR data').

ABSTRACT
 Background Systemic sclerosis (SSc) is an autoimmune condition characterized by fibrosis of skin and internal organs.Interstitial lung disease (SSc-ILD) is a major problem in SSc.In early disease, interleukin-6 may play a crucial role and may offer a potential target for disease modifying interventions.

 Main research question
To investigate the pathophysiological role of IL-6 in SSc-associated ILD  Design (including population, confounders/outcomes) Cross-sectional and experimental

 Expected results
With the current approach we aim to demonstrate a central role of Il-6 in early SSc-ILD.If our hypothesis proves to be valid, it will pave the way for a clinical interventional study in patients with early SSc-ILD.

BACKGROUND  Introduction and rationale
Systemic sclerosis (SSc) is an autoimmune condition characterized by fibrosis of skin and internal organs.In SSc, fibroblasts play a central role in the biology of the disease by interacting with endothelial cells and leukocytes in a complex biological network involving cytokines and adhesion molecules, resulting in excess deposition of extracellular matrix proteins and in tissue stiffening.Interstitial lung disease (SSc-ILD) is a major problem in SSc, since it accounts for about one-third of deaths and no disease modifying treatment is currently available.Recent reports have shown that early phases of SSc-ILD are associated with more pronounced inflammation, which may change to a more pro-fibrotic phenotype leading to irreversible fibrosis in later stages of disease.
Currently, only patients with extensive lung disease who have a poor prognosis and progressive fibrotic course qualify for treatment.First-line treatment options are limited to cyclophosphamide or Mycofenolate Mofetil (MMF).Due to adverse side effects and absence of studies in early SSc-ILD, these therapies are reserved for those patients with severe disease.We hypothesize that early treatment of the inflammatory phase in SSc-ILD would be favorable in preventing progression to irreversible lung fibrosis with interventions that targeted antiinflammatory effects and had limited side effects.Interleukin-6 (IL-6) has been shown to play an important role in SSc by regulating the function of immune and non-immune cells.IL-6 is known to induce TGFβ production and to enhance TGFβsignaling in dermal and cardiac fibroblasts.Conversely, TGFβ regulates the expression of IL-6 by lung fibroblasts and airway smooth muscle cells.We have recently shown that dermal fibroblast are capable of producing extremely high levels of IL-6 locally after stimulation.Dermal fibroblasts from patients with SSc express increased levels of IL-6 and an increased serum IL-6 levels predict higher mortality risk, worse skin involvement and increased pulmonary decline in SSc.In congruence with fibroblasts, alveolar macrophages obtained from SSc-ILD patients also appear to produce IL-6 at extremely high levels after GM-CSF stimulation.Elevated serum levels of IL-6 are associated with early disease.Importantly, IL-6 is only predictive of ILD outcome in patients with early disease, where it is not reflective in more progressed stages.Recently, a profound impact of IL-6R blockade on the activated fibroblast phenotype was shown using explant dermal fibroblast cultures from SSc patients before and after treatment.These data underline the importance of IL-6 in the early inflammatory phases of SSc and could be a novel target for early treatment of SSc-ILD.

Research question
We hypothesize that IL-6 plays a major role in early phase of the development of interstitial lung disease (ILD) in patients with Systemic Sclerosis (SSc).Since CRP levels in SSc patients with limited disease are generally only slightly elevated, the role of IL-6 is thought to be a local one, acting within the target tissue of the lung.In order to establish this role, several objectives need to be addressed.

Objectives:
1. to assess upregulation of inflammatory markers, including IL-6, in histological samples from patients with SSc-ILD 2. to assess the pro-inflammatory signature of lung fibroblasts obtained from lung biopsies and their IL-6 production after stimulation and whether this signature is influenced favorably by blockage of the IL-6 pathway.

Description study design
Design: This is a proof of concept study to establish the role of IL-6 in SSc-ILD, for which lung tissue have been obtained from waste material obtained from clinical practice including lung-explants following lung transplantation, obduction, and diagnostic procedures (VATS of open lung biopsies) for SSc-ILD.In vitro experiments on fibroblasts isolated from lung biopsies obtained for clinical practice.In vitro/ex vivo studies: Fibroblast will be sourced from BAL and biopsies.This has previously been shown to be feasible in material from patients with various types of interstitial lung diseases [Lehtonen 2014].

Measures:
Primary human lung fibroblasts will be stimulated with TGF-β to study -Profibrotic signature: Connective Tissue Growth Factor, Col1, αSMA by RNA expression and protein production.-IL-6 production -Migratory capacity and contractility activity by Using wound healing (scratch assays) and transwell expt for migration/proliferation. Collagen gel contraction assays to assess fibroblast contractility.-RNA profiling before and after stimulation with TGF -Culture of lung fibroblast obtained from bronchus brushes and bronchoalveolar lavage (BAL) from patients with SSc-ILD and effect of systemic treatment on profibrotic signature, IL-6 production and RNA profiling.o Total number of UMCG patients who will not be asked informed consent for screening: 25 ☒ Data/biomaterials will be obtained from an already existing internal or external (UMCG) bio-or databank (see Section 1. Study organization).Biobank Pathologie ☐ Data/biomaterials will be obtained from a previous study ('FAIR data' -internal/external; see Section 1. Study organization).<text>

5.4.2
Prospective study ☒ Not applicable (see section 5.2.2) 5.4.3Objection (Registry) in case one or more participants will not be asked informed consent, the objection registry will be checked for these participants and the data from those who objected will be excluded from the analyses.yes 5.4.4Informed consent (IC): access to identifiable participant data in case one or more study team members will have access to direct/indirect identifiable participant data, informed consent will be/has been obtained for this access.NA

IC: Collaboration with commercial parties
In case of collaboration with commercial/profit organizations, informed consent will be/has been obtained for this type of collaboration NA 5.4.6 IC: Linking with other registries In case the data will be linked with other registries, informed consent will be/has been obtained for this linkage(s) NA

IC: Incidental findings
In case there is a risk of incidental findings, informed consent will be/has been obtained to return findings to the participant NA 5.4.8IC: FAIR Data In case data collected for the present study will be shared for future studies, informed consent will be obtained for this NA 5.4.9IC: other aspects NA 5.4.10Withdrawal  Can participants withdraw informed consent before publication and will all data/ biomaterials of that participant be destroyed  Does the participant information letter contain information on how to withdraw NA NA No IC will be obtained, UMCG objection registry will be checked.

Research Data Management Plan (RDMP)
In this study the data will be collected, processed, and archived in accordance with the General Data Protection Regulation (GDPR) and the FAIR (Findable, Accessible, Interoperable, Reusable) principles under the responsibility of the Principal Investigator.A research data management plan (RDMP) will be drawn up to describe the further operational details and procedures.
X the RDMP section below is completed ⎕ a separate RDMP document will be attached to this protocol 5.5.1 Data collection  Only essential baseline characteristics and data required to answer the research question(s) will be collected.yes  Tooling (eg.software and procedures) used for collecting, processing, analysing, and storing data will be compliant with the UMCG policy and Standard Operating Procedures in the UMCG Research Toolbox.yes 5.5.2Anonymization and pseudonymization  Data will be anonymised during data collection (i.e.data cannot be linked back to the participant) Yes  Data will be pseudonymized by use of a code list during data collection.yes  Indirect and direct identifiable information collected will be minimized and only collected for the purpose of this study yes  Direct identifiable information will be stored separately from pseudonymized data in an electronic file

Management of biomaterials
Will biomaterials be collected, processed, analyzed and/or stored for the purpose of this study No skip section 5.6 <text> 5.6.1 Retrospective study (see sections 1, 5.2.2, and 5.4.1)If biomaterials will be used from a secondary/further use biobank that has not been approved by the Board of Directors of the UMCG, how will be prohibited that biomaterials necessary for future diagnostic/treatment purposes will be used in the present study.NA ☒ <text> 5.6.2Biomaterials collection  Only biomaterials required to answer the research question(s) will be collected yes/no  What biomaterials will be collected <text>  How will the biomaterials be collected and processed <text> 5.6.3Pseudonymization and access to biomaterials  Does the storage unit of the biomaterials comprise information that the participant (in)directly identifies, other than the participant's number and / or the sample number.
yes/no <if yes, explain>  Biomaterials can only be accessed by the Principal Investigator and study delegates after authorization by the Principal Investigator yes/no <if no, explain> 5.6.4Sharing of biomaterials (during and after study completion) In case biomaterials (and data) will leave the UMCG, will you contact the loket Contract Research to arrange the proper contracts?(Loket_Contract_Research@umcg.nl)NA/yes/no <if no, explain> 5.6.5 Biomaterials storage (during and after study completion)  Where and how will the biomaterials be stored <text>  Biomaterials will be stored 15 years after the study is completed yes/no <If no, explain and give number of years>  What will be done with the remaining biomaterials after study completion (eg.destroyed, returned to biobank/previous study, stored) <text> 5.6.

Data analysis
 Power analysis NA  Statistical analysis Statistical analysis will be carried out using IBM SPSS Statistics version 23 and will be largely descriptional.If applicable, effect sizes will be calculated which will help calculating sample sizes for future studies.Non-parametric statistical test will be performed due to small number.Differences between groups were tested with Fisher's exact test.Spearman's rho will be applied for association.Values of p<0.05 are considered statistically significant.Data will be described as median (IQR) or number (percentage).

Participant information after the study
Will participants be informed about the study results no

Research revenue
In case the study will result in revenues (e.g. as a result of the use of data/biomaterials or successful licensing of intellectual property or manufactured products), will you contact the loket Contract Research to arrange the proper contracts?
NA <if no, explain> Describe what will be done with the revenues.<text>

NA
Corresponding researcher UMCG DJ Mulder, MD, PhD, internist-vascular specialist, dept.Internal Medicine/Vascular Medicine, University Medical Center Groningen, University of Groningen, University of Groningen, Groningen, The Netherlands.Name bio-or databank and bankmanager J.J. Duker Biobank Pathologie Department of Pathology and Medical Biology, Medical Biology Section, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.j.j.duker@umcg.nl☒approved by the Board of Directors UMCG ☐ not approved by the Board of Directors UMCG Name previous study ('FAIR data') and (principal) investigator NA


Data access (during the study)  Direct identifiable information can only be accessed by the Principal Investigator and study delegates after authorization by the Principal Investigator.yes  Pseudonymized/anonymized data can only be accessed by the Principal Investigator and study delegates after authorization by the Principal Investigator.yesData roles, responsibilities, access and authorization -during the study and after study completion -will be managed and documented (e.g. in the RDMP, on study delegation log).yes 5.5.4Data sharing (during and after study completion) In case data (and biomaterials) will leave the UMCG, will you contact the loket Contract Research to arrange the proper contracts?(Loket_Contract_Research@umcg.nl)yes 5.5.5 Data storage (during and after study completion)  Digital data will be archived on the UMCG network complying with strict UMCG security and back-up policy.yesPaper source data and study files will be archived within the UMCG facilities.yes  Source data, study files and digital data will be stored 15 years after the study is completed.yes 5.5.6 Data re-use and access after completion of the present study ('FAIR data') NA ☒ Diagnosis of Systemic Sclerosis, as determined by 2013 ACR/EULAR criteria, as established by the treating physician. A formal diagnosis of SSc-ILD based of typical lung function or HRCT abnormalities as established by the treating physician. Exclusion criteria • ILD as a result of other connective tissue disease, idiopathic pulmonary fibrosis • Other lung diseases associated with inflammation or fibrosis, including COPD, asthma, cystic fibrosis, lung cancer.
no 5.4 Recruitment and informed consent/objection 5.4.1 Retrospective study (tick all that apply)☐ Not applicable (see section 5.2.2) ☒ Data will be copied from (electronic) patient records (e.g.'nieuw EPD' UMCG)  A list of specimen potentially suitable for the study will be supplied to the researcher.In the patient record, data will be identified (if available) which are needed for interpretation of the study results, and copied to a case report form, these include: o Age specimen was obtained, sex, race, body weight, length o Clinical characteristics of SSc, including SSc subtype (diffuse 6 Biomaterials re-use and access after completion of the present study ('FAIR data') Biomaterials will be made findable by including the description of the study (and type of biomaterials in the UMCG FAIR data catalogue and other discipline specific catalogue(s).If participants are patients: Can be deviated from the standard care / diagnostic procedures (e.g. can medical treatment be postponed or limited) Procedure to assess if a finding should be returned to the participant, or not <text> o Procedure to inform the participant <text> 